1. Field of the Invention
This invention relates to the field of modified antisense oligonucleotides for in vitro and in vivo research, diagnostic, and therapeutic purposes.
2. Summary of the Related Art
Since Zamecnik and Stephenson (Proc. Natl. Acad. Sci. USA 75, 280 (1978)) first demonstrated virus replication inhibition by synthetic oligonucleotides, great interest has been generated in oligonucleotides as research, diagnostic, and therapeutic agents. In recent years the development of oligonucleotides as therapeutic agents and as agents of gene expression modulation has gained great momentum. The greatest development has been in the use of so-called antisense oligonucleotides, which form Watson-Crick duplexes with target mRNAs. Agrawal (Trends in Biotechnology 10, 152 (1992)) extensively reviews the development of antisense oligonucleotides as antiviral agents.
Great strides have been made in the development of antisense oligonucleotides for a variety of purposes. The use of antisense oligonucleotides as useful research tools to modulate gene expression is manifested, for example, by Holt et al. (Mol. Cell Biol. 8, 963 (1988)), who employed an antisense oligomer to inhibit HL-60 c-myc expression in order to study the role of a nuclear protooncogene in the regulation of cell growth and differentiation.
Other studies have demonstrated the utility of antisense oligonucleotides for research and therapeutic use. Antisense Therapeutics (S. Agrawal, Ed., Humana Press, Totowa, N.J., 1996); Thoung and Helene, Angew. Chem. Int. Ed. Eng. 32, 666 (1993); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993).
Rapid degradation of "natural" phosphodiester backbone oligonucleotides by cellular nucleases (Sands et al., Mol. Pharmacol. 45, 932 (1994); Agrawal et al., Clin. Pharmacokinet. 28, 7 (1995)) necessitated chemical modifications of the phosphodiester backbone. Agrawal and Iyer (Current Opin. Biotech. 6, 12 (1995)) review a number of these approaches. See also Uhlmann and Peyman (Chem. Rev. 90, 543 (1990)). Several chemically modified oligonucleotides such as methylphosphonate (Smith et al., Proc. Natl. Acad. Sci. USA 83, 2787-2791 (1986); Sarin et al., Proc. Natl. Acad. Sci. USA 85, 7448-7451 (1988); Matsukura et al., Proc. Natl. Acad. Sci. USA 84, 7706-7710 (1987)), phosphorothioate (Id.), and phosphoramidate (Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988)) oligonucleotides show higher stability against nucleases. Many of these modifications have been tested against several disease targets in vitro and in vivo. (Agrawal, Trends in Biotech., supra.; Agrawal and Iyer, Curr. Opn. Biotech. 6, 12-19 (1995)) Barker et al. (Proc. Natl. Acad. Sci. USA 93, 514 (1996)), for example, demonstrated successful inhibition of several genes of Plasmodium falciparum (which is responsible for malaria) in whole cell erythrocytes. They found that phosphorothioate antisense oligonucleotides can and do enter parasitized erythrocytes and inhibit expression of a number of genes essential to the reproduction and growth of the parasite.
Offensperger et al. (EMBO J. 12, 1257 (1993)) demonstrated complete inhibition of duck hepatitis B virus (DHBV) in DHBV-infected Peking ducks in vivo.
Simons et al. (Nature 359, 67 (1992)) reported that a phosphorothioate antisense c-myb 18-mer locally delivered to a rat with an injured left common carotid artery suppressed c-myb mRNA concentrations 2 weeks after injury and blocked the accumulation of intimal smooth muscle cells.
Ratajczak et al. (Proc. Natl. Acad. Sci. U.S.A. 89, 11823 (1992)) reported that 24-mer phosphorothioate oligonucleotides targeted to the human c-myb mRNA were infused, through a mini-osmotic pump, into scid mice bearing the human K562 chromic myeloid leukemia cell line. Mean survival times of the mice treated with the antisense oligonucleotide were six- to eightfold longer than those of mice untreated or treated with the sense controls.
Kitajima et al. (Science 258, 1792 (1992)) reported that after injecting IP 3'-phosphorothioate modified phosphodiester chimeric oligonucleotides that were complementary to the initiation codon region of the NF-.kappa.B mRNA (p65), they observed complete tumor involution in 13 out of 13 antisense-treated transgenic mice having that gene. Untreated or sense-treated mice died by 12 weeks, whereas the treated animals had no recurrence for at least 5 months.
Nesterova and Cho-Chung (Nature Medicine 1, 528 (1995)) reported in vivo tumor growth suppression in nude mice bearing LS-174T human colon carcinoma with antisense oligonucleotides targeted to the RI.sub.60, subunit of cAMP-dependent protein kinase type I nucleic acid.
The phosphorothioate oligonucleotides advanced to human clinical trials because of their desirable properties observed in in vitro and in vivo studies. (Zhang et al., Clin. Pharmacol. Ther. 58, 44-53 (1995); Crooke et al., Clin. Pharmacol. Ther. 56, 641-646 (1994); Bayever et al., Antisense Res. Dev. 3, 383-390 (1993)). In order to improve pharmacokinetic properties of antisense phosphorothioate oligodeoxyribonucleotides, mixed backbone oligonucleotides (MBOs) have been designed that contain at least two different chemical modifications. Recent studies on MBOs such as hybrids, chimeras, etc., suggest that MBOs have improved pharmacokinetic properties and reduced toxicity with retention of comparable efficacy to phosphorothioate oligodeoxyribonucleotides. Zhang et al., Biochem. Pharmacol. 50, 545 (1995).
Most of the modifications currently explored for antisense purposes use the commonly occurring 3'-5' internucleotide linkage. In addition to the predominant 3'-5' internucleotide linkage, a less abundant 2'-5'-internucleotide linkage is formed in cells treated with interferon (Kerr and Brown, Proc. Natl. Acad. Sci. USA 75, 256 (1978); Lesiak et al., J Biol. Chem. 258, 13082 (1983)) and during intron splicing (Padget et al., Science 225, 898 (1984)).
Michelson and Monny (Biochim. Biophys. Acta 149, 107 (1967)) demonstrated that short 2'-5'-oligoadenylates form stable complexes with poly rU, although with lower T.sub.m 's than their 3'-5' counterparts. Ts'o et al. (Biochem. 8, 997 (1969)), Tazawa et al. (Biochem. 8, 3499 (1970)), and Kondo et al. (Biochem. 9, 3479 (1970)) studied 2'-5'-linked r(ApA) and its binding to poly rU. Westheirner (Acc. Chem. Res. 1, 70 (1968)) demonstrated stability of 2'-5'-dimers and trimers to a few hydrolytic nucleases. Although the formation of 2'-5'-linkage is preferred over a 3'-5'-linkage under simulated prebiotic conditions (22,23), nature's selection of the 3'-5'-linkage over the 2'-5'-linkage to preserve genetic material has long been debated. Hashimoto and Switzer, J. Am. Chem. Soc. 114, 6255-6256 (1992); Jin et al., Proc. Natl. Acad. Sci. USA 90, 10568-10572 (1993); Usher and McHale, Proc. Natl. Acad. Sci. USA 73, 1149-1153 (1976).
More recently, Dougherty et al. (J. Am. Chem. Soc. 114, 6254 (1992)) and Hashimoto and Switzer (J. Am. Chem. Soc. 114, 6255 (1992)) reported preparation and hybridization properties of 2'-5'-DNA. Kierzek et al. (Nucleic Acids Res. 20, 1685 (1992)) presented a similar study of 2'-5'-RNA. None of the foregoing studies were directed to antisense applications, however, as binding to 3'-5' RNA or DNA was not considered.
The utility of 2'-5'-linked oligonucleotides for antisense use has not been explored extensively, although a possible application has been suggested. Giannaris and Damha (Nucleic Acids Res. 21, 4742 (1993)) studied the hybridization characteristics of 2'-5'-RNA and hybrids of 2'-5'-RNA and 3'-5'-RNA with natural (i. e., 3'-5') single-stranded RNA and DNA. They found that introduction of 2'-5' internucleotide linkages decreases the T.sub.m of the duplexes formed between the 2'-5'-linkage-containing oligonucleotide and its target. They also observed that the 2'-5'-linkage-containing oligonucleotide selectively hybridized to RNA targets over DNA targets.
Seki et al. (Nucleic Acids Symp. Series 29, 71 (1993)) and Sawai et al. (J. Biomol. Structure Dyn. 13, 104 (1996)) reported on the hybridization affinities of 2'-5 '-linked oligo(rA) and oligo(rU) to each other and to 3'-5'-linked oligo(rA) and oligo(rU). They observed that a mixture of 2'-5' oligo(rA) and oligo(rU) formed double and triple helices in the same manner as a mixture 3'-5'-linked oligo(rA) and oligo(rU), but with lower T.sub.m. The mixture of 2'-5'-linked oligo(rA) and oligo(rU) formed only a duplex at a much lower T.sub.m. Thermodynamic studies showed that 3'-5'-linked oligoRNA possess more favorable base stacking and base pairing interactions compared to 2'-5'-linked RNA.
Alul (U.S. Pat. No. 5,532,130) disclosed 2'-5'-linked oligonucleotide-3'-deoxyribonucleotides for hybridizing to complementary RNA. They observed that such oligonucleotides selectively hybridize to RNA rather than DNA.
Despite the advances made, however, there is still a continuing interest in developing new and useful modifications to improve antisense oligonucleotide efficacy.